System, method, and product for generating patterned illumination

ABSTRACT

An embodiment of a method for generating an interference pattern at a probe array is described that comprises directing light at a first waveguide and second waveguide, wherein the first and second waveguides are positioned adjacent to each other and the output from the first and second waveguides produce an interference pattern; and directing the interference pattern at the probe array.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/777,914, filed on Jul. 13, 2007, which claims priority to ProvisionalApplication Ser. No. 60/820,002 filed on Jul. 21, 2006, which is hereinincorporated by reference in its entirety for all purposes.

BACKGROUND

1. Field of the Invention

The present invention relates to systems and methods for examiningbiological material. In particular, the invention relates to a system,method, and product for generating an interference pattern at thefunctional surface of a probe array comprising small dimension probefeatures each having thousands of probe molecules disposed therein. Forexample, it may be advantageous for systems using biological probefeatures 1 μm in dimension or smaller to employ specialized methods,optical elements, and/or detection schemes to achieve desired levels ofresolution. One such method may be referred to as Patterned Illuminationor Patterned Excitation that include generating an interference patternof excitation light at the surface of a probe array and detectingdisturbances in the pattern associated with the probe features. Opticalsystems have been described to generate interference patterns thatinclude complicated arrays of mirrors, beam splitters, and othercomponents for controlling the pattern including the orientationrelative to the probe array, phase, and other characteristics. Thepresently described invention includes a less complicated, expensive,and sensitive means for generating the interference pattern that employsa pair of adjacent waveguides.

2. Related Art

Synthesized nucleic acid probe arrays, such as Affymetrix GeneChip®probe arrays, and spotted probe arrays, have been used to generateunprecedented amounts of information about biological systems. Forexample, the GeneChip® Human Genome U133 Plus 2.0 Array available fromAffymetrix, Inc. of Santa Clara, Calif., is comprised of one microarraycontaining 1,300,000 oligonucleotide features covering more than 47,000transcripts and variants that include 38,500 well characterized humangenes. Other examples of GeneChip® arrays are targeted to provide dataaimed at different areas of specialization. Examples of specialized usesinclude analysis of Single Nucleotide Polymorphisms (SNPs) provided bythe GeneChip® Human Mapping 10K, 100K, 500K, or 6.0 Arrays, or analysisof alternative splicing events provided by the GeneChip® Human Exon 1.0ST Array. Analysis of data from such microarrays may lead to thedevelopment of new drugs and new diagnostic tools.

SUMMARY OF THE INVENTION

Systems, methods, and products to address these and other needs aredescribed herein with respect to illustrative, non-limiting,implementations. Various alternatives, modifications and equivalents arepossible. For example, certain systems, methods, and computer softwareproducts are described herein using exemplary implementations foranalyzing data from arrays of biological materials, in particular inrelation to data from Affymetrix® GeneChip® probe arrays. However, thesesystems, methods, and products may be applied with respect to many othertypes of probe arrays and, more generally, with respect to numerousparallel biological assays produced in accordance with otherconventional technologies and/or produced in accordance with techniquesthat may be developed in the future. For example, the systems, methods,and products described herein may be applied to parallel assays ofnucleic acids, PCR products generated from cDNA clones, proteins,antibodies, or many other biological materials. These materials may bedisposed on slides (as typically used for spotted arrays), on substratesemployed for GeneChip® arrays, or on beads, optical fibers, or othersubstrates or media, which may include polymeric coatings or otherlayers on top of slides or other substrates. Moreover, the probes neednot be immobilized in or on a substrate, and, if immobilized, need notbe disposed in regular patterns or arrays. For convenience, the term“probe array” will generally be used broadly hereafter to refer to allof these types of arrays and parallel biological assays.

An embodiment of a method for generating an interference pattern at aprobe array is described that comprises directing light at a firstwaveguide and second waveguide, wherein the first and second waveguidesare positioned adjacent to each other and the output from the first andsecond waveguides produce an interference pattern; and directing theinterference pattern at the probe array.

One embodiment of the present invention is a method for generating aninterference pattern to interrogate a surface of a support comprisingdirecting light at a first waveguide, directing light at a secondwaveguide, wherein the first and second waveguides are positioned in anoperative relationship so as to produce an interference pattern,directing the interference pattern at a surface of a support, anddetecting signals from the surface of the support. The probe array has abiopolymer affixed to the surface of a support selected from the groupconsisting of nucleic acids, oligonucleotides, amino acids, proteins,peptides, hormones, oligosaccharides, lipids, glycolipids,lipopolysaccharides, phospholipids, inverted nucleotides, peptidenucleic acids, and Meta-DNA. The detector comprises a CCD, EMCCD, CMOS,APS, or PMT. Preferably, the waveguides are optical fibers orrectangular waveguides. Systems containing the above features are alsopart of the present invention.

The above embodiments and implementations are not necessarily inclusiveor exclusive of each other and may be combined in any manner that isnon-conflicting and otherwise possible, whether they be presented inassociation with a same, or a different, embodiment or implementation.The description of one embodiment or implementation is not intended tobe limiting with respect to other embodiments and/or implementations.Also, any one or more function, step, operation, or technique describedelsewhere in this specification may, in alternative implementations, becombined with any one or more function, step, operation, or techniquedescribed in the summary. Thus, the above embodiment and implementationsare illustrative rather than limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further features will be more clearly appreciated from thefollowing detailed description when taken in conjunction with theaccompanying drawings. In the drawings, like reference numerals indicatelike structures or method steps and the leftmost digit of a referencenumeral indicates the number of the figure in which the referencedelement first appears (for example, the element 160 appears first inFIG. 1). In functional block diagrams, rectangles generally indicatefunctional elements and parallelograms generally indicate data. Inmethod flow charts, rectangles generally indicate method steps anddiamond shapes generally indicate decision elements. All of theseconventions, however, are intended to be typical or illustrative, ratherthan limiting.

FIG. 1 is a functional block diagram of one embodiment of a computer anda server enabled to communicate over a network, as well as a probe arrayand probe array instruments;

FIG. 2 is a functional block diagram of one embodiment of the computersystem of FIG. 1, including a display device that presents a graphicaluser interface to a user;

FIG. 3 is a functional block diagram of one embodiment of the server ofFIG. 1, where the server comprises an executable version of aninstrument control and image processing application and an analysisapplication;

FIG. 4A is a simplified graphical representation of one embodiment of awaveguide and dispersion cone;

FIG. 4B is a simplified graphical representation of two adjacentwaveguides and overlapping dispersion cones that create an interferencepattern; and

FIG. 5 is a simplified graphical representation of one embodiment of theinterference pattern of FIG. 4B.

DETAILED DESCRIPTION

The present invention has many aspects and preferred embodiments. Oneparticular embodiment is a waveguided structured illumination technique.This technique uses two adjacent waveguides to create an interferencepattern in order to generate a phase-shift-able and rotate-ablestructured illumination pattern for high resolution imaging for use inmicroarray imaging and other fields. One preferred embodiment of theinvention rotates the waveguide assembly about the beam axis to alterthe angles of the interference pattern with respect to the microarray.

This embodiment of the invention uses a variable index of refractioncladding or similar technology (including labeled biological surface infarfield's picture) on at least one surface of the optical wave guide totranslate the phase of the interference pattern.

The rotation of the waveguide assembly is useful in one embodiment ofthe invention. Rotation enables the system to mimic the capability of amulti mirror system with alternative and potentially advantageoushardware. Repeated fluorescent acquisition could use CCD imagers as oneexample.

High density microarray technology has revolutionized biologicalanalyses. It has been extensively used for clinical diagnostics,toxicology, genomics, drug discovery, environmental monitoring,genotyping and many other fields (Fodor, S. P.; Read, J. L.; Pirrung, M.C.; Stryer, L.; Lu, A. T.; Solas, D. Light-directed, spatiallyaddressable parallel chemical synthesis, Science 251(4995), 767-73,1991; Fodor, S. P.; Rava, R. P.; Huang, X. C.; Pease, A. C.; Holmes, C.P.; Adams, C. L., Multiplexed biochemical assays with biological chips,Nature 364(6437), 555-6, 1993; Pease, A. C.; Solas, D.; Sullivan, E. J.;Cronin, M. T.; Holmes, C. P.; Fodor, S. P., Light-generatedoligonucleotide arrays for rapid DNA sequence analysis, Proceedings ofthe National Academy of Sciences of the United States of America 91(11),5022-6, 1994). Fluorescence labels are frequently used for microarraydetection. A variety of image acquisition devices, such as CCD (chargecoupled device), are used for detecting binding patterns.

In one aspect of the invention, a method for microarray detection isprovided. In one aspect of the invention, methods and devices areprovided for microarray detection using a series of structured,textured, or patterned excitation (referred herein as patternedexcitation) images to achieve subpixel resolution in detecting probeintensities. The microarray can be a nucleic acid probe array such as aspotted array (e.g., with cDNA or short oligonucleotide probes), highdensity in situ synthesized arrays (such as the GeneChip® high densityprobe arrays manufactured by Affymetrix, Inc., Santa Clara, Calif.). Themicroarrays can also be protein or peptide arrays. Typically, thedensity of the microarrays is higher than 500, 5000, 50000, or 500,000different probes per cm². The feature size of the probes (synthesis areaor immobilization area) is typically smaller than 500, 150, 25, 9, 5, 3or 1 μm².

In one aspect of the invention, a method for microarray analysis isprovided. The method includes obtaining a series of fluorescent imagesof a microarray, where the fluorescent signals reflect binding betweentargets and probes, and where each of the images is obtained with adifferent excitation pattern; and analyzing the images using calibratedinformation about the different excitation patterns and probe featureinformation to obtain intensities for each of the probes. The methodtypically includes generating a composite image where the compositeimage has a higher resolution than those of the fluorescent images. Thedifferent excitation patterns are generated by translating excitationpatterns and different laser beam pair configurations.

One of skill in the art would appreciate that many different methods maybe used to generate light patterns that can be used with patternedexcitation. The methods of the invention are not limited to anyparticular methods for generating light patterns.

Typically, the images are obtained using a photo detector array.However, a single photo detector, such as a PMT may be used in someembodiments. The photo detector array can be a charge coupled device(CCD) such as an electron multiplication CCD (EMCCD). CMOS imagers suchas an Active Pixel Sensor (APS) may also be used.

Information about different excitation patterns may include spatialfrequency information such as orientation and spacing between adjacentpeak intensities. In some embodiments, the analyzing steps includeextracting cosine parameters to obtain I_(DC), (DC component ofintensity values), I_(AC) (AC component of intensity values), and φ(timing information, where the peak intensity appears) of pixelintensities. In a preferred embodiment, the analyzing step includesconstructing a system of linear equations that relate the pixelintensities, subpixel weighting functions, and unknown subpixelintensities. For example, the linear equations may be as follows:

${{b^{i}(k)} = {\sum\limits_{m}{\sum\limits_{n}{{W^{i}\left( {{m.},n,k} \right)}{I^{i}\left( {m,n} \right)}}}}},$

where I^(i) (m,n) is the unknown subpixel intensities; W^(i)(m,n, k) isthe weighting function within i-th pixel for k-th frame at a subpixellocation (m,n); and b^(i)(k) is the sequence of gray intensity values ofi-th pixel. The equations may be solved to obtain subpixel intensities.

The weighting function W^(i)(m,n, k) can be calculated, for example,using pattern calibration parameters as:E_(CD)+E_(AC)·cos(k_(x)·x+k_(y)·y+φ), where E_(DC) and E_(AC) are DC andAC components of the pattern intensities, respectively; k_(x) and k_(y)are x and y components of the pattern spatial frequency, respectfully;and the φ represents subpixel position of the pattern. Alternatively,the weighting function W^(i)(m,n, k) is calculated by solving theequation

${b^{i}(k)} = {\sum\limits_{m}{\sum\limits_{n}{{W^{i}\left( {{m.},n,k} \right)}{I^{i}\left( {m,n} \right)}}}}$

using data obtained with reference samples with known subpixelintensities.

In another aspect of the invention, the intensity values are estimatedusing optimization methods. In some embodiments, the subpixelintensities are estimated with probe feature information as constraints.For example, the regularity of the probe features is used asconstraints. The dynamic range the probe intensities can also be used.One particularly preferred method is to minimize

${{{b^{i}(k)} = {\sum\limits_{m}{\sum\limits_{n}{{W^{i}\left( {m,n,k} \right)}{I^{i}\left( {m,n} \right)}}}}}}^{2}.$

Liner programming is a preferred method for estimating the intensityvalues.

In another aspect of the invention, computer software products formicroarray analysis are provided. Such products typically have acomputer readable medium containing computer-executable instructions forperforming the method of the invention. The software code structurestypically include components for executing various steps of the methods.

In yet another aspect of the invention, a system for performing themethods of the invention is provided. Such a system typically includes acomputer processor; and a memory coupled with the processor, the memorystoring a plurality of machine instructions that cause the processor toperform logical steps of the methods of the invention.

A system of the invention may also include a patterned excitation unitthat has a excitation source beam, a pattern generator, image optics, animager (such as a CCD). The computer unit may control the patternedexcitation unit and receive the data from the image. The computer unitmay contain computer software codes that perform pattern calibration,cosine parameter extraction, feature extraction, etc.

a) GENERAL

The present invention has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a patent, application, or other reference iscited or repeated below, it should be understood that it is incorporatedby reference in its entirety for all purposes as well as for theproposition that is recited.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

An individual is not limited to a human being but may also be otherorganisms including but not limited to mammals, plants, bacteria, orcells derived from any of the above.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, N.Y., Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The present invention can employ solid substrates, including arrays insome preferred embodiments. Methods and techniques applicable to polymer(including protein) array synthesis have been described in U.S. Ser. No.09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867,5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839,5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832,5,856,101, 5,858,659, 5,936,324, 5,945,334, 5,968,740, 5,974,164,5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555,6,136,269, 6,269,846 and 6,428,752, in PCT Applications Nos.PCT/US99/00730 (International Publication Number WO 99/36760) andPCT/US01/04285 (International Publication Number WO 01/58593), which areall incorporated herein by reference in their entirety for all purposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098. Nucleic acid arrays are described in many ofthe above patents, but the same techniques are applied to polypeptidearrays. Nucleic acid arrays that are useful in the present inventioninclude those that are commercially available from Affymetrix (SantaClara, Calif.) under the brand name GeneChip®. Example arrays are shownon the website at affymetrix.com.

The present invention also contemplates many uses for polymers attachedto solid substrates. These uses include gene expression monitoring,profiling, library screening, genotyping and diagnostics. Geneexpression monitoring and profiling methods can be shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248and 6,309,822. Genotyping and uses therefore are shown in U.S. Ser. Nos.10/442,021, 10/013,598 (U.S. Patent Application Publication20030036069), and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659,6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses are embodiedin U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and6,197,506.

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with genotyping,the genomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. See, e.g., PCR Technology: Principles andApplications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188,and 5,333,675, and each of which is incorporated herein by reference intheir entireties for all purposes. The sample may be amplified on thearray. See, for example, U.S. Pat. No. 6,300,070 and U.S. Ser. No.09/513,300, which are incorporated herein by reference.

Other suitable amplification methods include the ligase chain reaction(LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al.,Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be used aredescribed in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S.Ser. No. 09/854,317, each of which is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 andU.S. Ser. Nos. 09/916,135, 09/920,491 (U.S. Patent ApplicationPublication 20030096235), 09/910,292 (U.S. Patent ApplicationPublication 20030082543), and 10/013,598.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. ColdSpring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology,Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc.,San Diego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described in U.S. Pat. Nos. 5,871,928,5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which areincorporated herein by reference

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred embodiments. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Ser. No. 10/389,194 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. Nos. 10/389,194,10/913,102, 10/846,261, 11/260,617 and in PCT Application PCT/US99/06097(published as WO99/47964), each of which also is hereby incorporated byreference in its entirety for all purposes.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, e.g.Setubal and Meidanis et al., Introduction to Computational BiologyMethods (PWS Publishing Company, Boston, 1997); Salzberg, Searles,Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier,Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics Applicationin Biological Science and Medicine (CRC Press, London, 2000) andOuelette and Bzevanis Bioinformatics: A Practical Guide for Analysis ofGene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001). See U.S. Pat.No. 6,420,108.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing genetic information over networks such asthe Internet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559 (UnitedStates Publication No. 20020183936), 10/065,856, 10/065,868, 10/328,818,10/328,872, 10/423,403, and 60/482,389.

b) DEFINITIONS

The term “array” as used herein refers to an intentionally createdcollection of molecules which can be prepared either synthetically orbiosynthetically. The molecules in the array can be identical ordifferent from each other. The array can assume a variety of formats,e.g., libraries of soluble molecules; libraries of compounds tethered toresin beads, silica chips, or other solid supports.

The term “biomonomer” as used herein refers to a single unit ofbiopolymer, which can be linked with the same or other biomonomers toform a biopolymer (for example, a single amino acid or nucleotide withtwo linking groups one or both of which may have removable protectinggroups) or a single unit which is not part of a biopolymer. Thus, forexample, a nucleotide is a biomonomer within an oligonucleotidebiopolymer, and an amino acid is a biomonomer within a protein orpeptide biopolymer; avidin, biotin, antibodies, antibody fragments,etc., for example, are also biomonomers.

The term “biopolymer” or “biological polymer” as used herein is intendedto mean repeating units of biological or chemical moieties.Representative biopolymers include, but are not limited to, nucleicacids, oligonucleotides, amino acids, proteins, peptides, hormones,oligosaccharides, lipids, glycolipids, lipopolysaccharides,phospholipids, synthetic analogues of the foregoing, including, but notlimited to, inverted nucleotides, peptide nucleic acids, Meta-DNA, andcombinations of the above.

The term “biopolymer synthesis” as used herein is intended to encompassthe synthetic production, both organic and inorganic, of a biopolymer.Related to a biopolymer is a “biomonomer”.

The term “complementary” as used herein refers to the hybridization orbase pairing between nucleotides or nucleic acids, such as, forinstance, between the two strands of a double stranded DNA molecule orbetween an oligonucleotide primer and a primer binding site on a singlestranded nucleic acid to be sequenced or amplified. Complementarynucleotides are, generally, A and T (or A and U), or C and G. Two singlestranded RNA or DNA molecules are said to be complementary when thenucleotides of one strand, optimally aligned and compared and withappropriate nucleotide insertions or deletions, pair with at least about80% of the nucleotides of the other strand, usually at least about 90%to 95%, and more preferably from about 98 to 100%. Alternatively,complementarity exists when an RNA or DNA strand will hybridize underselective hybridization conditions to its complement. Typically,selective hybridization will occur when there is at least about 65%complementary over a stretch of at least 14 to 25 nucleotides,preferably at least about 75%, more preferably at least about 90%complementary. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984),incorporated herein by reference.

The term “combinatorial synthesis strategy” as used herein refers to acombinatorial synthesis strategy is an ordered strategy for parallelsynthesis of diverse polymer sequences by sequential addition ofreagents which may be represented by a reactant matrix and a switchmatrix, the product of which is a product matrix. A reactant matrix is a1 column by m row matrix of the building blocks to be added. The switchmatrix is all or a subset of the binary numbers, preferably ordered,between 1 and m arranged in columns. A “binary strategy” is one in whichat least two successive steps illuminate a portion, often half, of aregion of interest on the substrate. In a binary synthesis strategy, allpossible compounds which can be formed from an ordered set of reactantsare formed. In most preferred embodiments, binary synthesis refers to asynthesis strategy which also factors a previous addition step. Forexample, a strategy in which a switch matrix for a masking strategyhalves regions that were previously illuminated, illuminating about halfof the previously illuminated region and protecting the remaining half(while also protecting about half of previously protected regions andilluminating about half of previously protected regions). It will berecognized that binary rounds may be interspersed with non-binary roundsand that only a portion of a substrate may be subjected to a binaryscheme. A combinatorial “masking” strategy is a synthesis which useslight or other spatially selective deprotecting or activating agents toremove protecting groups from materials for addition of other materialssuch as amino acids.

The term “complex population or mixed population” as used herein refersto any sample containing both desired and undesired nucleic acids. As anon-limiting example, a complex population of nucleic acids may be totalgenomic DNA, total genomic RNA or a combination thereof. Moreover, acomplex population of nucleic acids may have been enriched for a givenpopulation but include other undesirable populations. For example, acomplex population of nucleic acids may be a sample which has beenenriched for desired messenger RNA (mRNA) sequences but still includessome undesired ribosomal RNA sequences (rRNA).

The term “effective amount” as used herein refers to an amountsufficient to induce a desired result.

The term “genome” as used herein is all the genetic material in thechromosomes of an organism. DNA derived from the genetic material in thechromosomes of a particular organism is genomic DNA. A genomic libraryis a collection of clones made from a set of randomly generatedoverlapping DNA fragments representing the entire genome of an organism.

The term “hybridization conditions” as used herein will typicallyinclude salt concentrations of less than about 1M, more usually lessthan about 500 mM and preferably less than about 200 mM. Hybridizationtemperatures can be as low as 5.degree. C., but are typically greaterthan 22.degree. C., more typically greater than about 30.degree. C., andpreferably in excess of about 37.degree. C. Longer fragments may requirehigher hybridization temperatures for specific hybridization. As otherfactors may affect the stringency of hybridization, including basecomposition and length of the complementary strands, presence of organicsolvents and extent of base mismatching, the combination of parametersis more important than the absolute measure of any one alone.

The term “hybridization” as used herein refers to the process in whichtwo single-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization.” Hybridizations are usually performed understringent conditions, for example, at a salt concentration of no morethan 1 M and a temperature of at least 25° C. For example, conditions of5×SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and atemperature of 25-30° C. are suitable for allele-specific probehybridizations. For stringent conditions, see, for example, Sambrook,Fritsche and Maniatis. “Molecular Cloning A laboratory Manual” 2^(nd)Ed. Cold Spring Harbor Press (1989) which is hereby incorporated byreference in its entirety for all purposes above.

Hybridizations, e.g., allele-specific probe hybridizations, aregenerally performed under stringent conditions. For example, conditionswhere the salt concentration is no more than about 1 Molar (M) and atemperature of at least 25 degrees-Celsius (C), e.g., 750 mM NaCl, 50 mMNaPhosphate, 5 mM EDTA, pH 7.4 (5×SSPE) and a temperature of from about25 to about 30° C.

The term “hybridization probes” as used herein are oligonucleotidescapable of binding in a base-specific manner to a complementary strandof nucleic acid. Such probes include peptide nucleic acids, as describedin Nielsen et al., Science 254, 1497-1500 (1991), and other nucleic acidanalogs and nucleic acid mimetics.

The term “hybridizing specifically to” as used herein refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence or sequences under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA.

The term “initiation biomonomer” or “initiator biomonomer” as usedherein is meant to indicate the first biomonomer which is covalentlyattached via reactive nucleophiles to the surface of the polymer, or thefirst biomonomer which is attached to a linker or spacer arm attached tothe polymer, the linker or spacer arm being attached to the polymer viareactive nucleophiles.

The term “isolated nucleic acid” as used herein mean an object speciesinvention that is the predominant species present (i.e., on a molarbasis it is more abundant than any other individual species in thecomposition). Preferably, an isolated nucleic acid comprises at leastabout 50, 80 or 90% (on a molar basis) of all macromolecular speciespresent. Most preferably, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods).

The term “ligand” as used herein refers to a molecule that is recognizedby a particular receptor. The agent bound by or reacting with a receptoris called a “ligand,” a term which is definitionally meaningful only interms of its counterpart receptor. The term “ligand” does not imply anyparticular molecular size or other structural or compositional featureother than that the substance in question is capable of binding orotherwise interacting with the receptor. Also, a ligand may serve eitheras the natural ligand to which the receptor binds, or as a functionalanalogue that may act as an agonist or antagonist. Examples of ligandsthat can be investigated by this invention include, but are notrestricted to, agonists and antagonists for cell membrane receptors,toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids,etc.), hormone receptors, peptides, enzymes, enzyme substrates,substrate analogs, transition state analogs, cofactors, drugs, proteins,and antibodies.

The term “linkage disequilibrium or allelic association” as used hereinrefers to the preferential association of a particular allele or geneticmarker with a specific allele, or genetic marker at a nearby chromosomallocation more frequently than expected by chance for any particularallele frequency in the population. For example, if locus X has allelesa and b, which occur equally frequently, and linked locus Y has allelesc and d, which occur equally frequently, one would expect thecombination ac to occur with a frequency of 0.25. If ac occurs morefrequently, then alleles a and c are in linkage disequilibrium. Linkagedisequilibrium may result from natural selection of certain combinationof alleles or because an allele has been introduced into a populationtoo recently to have reached equilibrium with linked alleles.

The term “mixed population” as used herein refers to a complexpopulation.

The term “monomer” as used herein refers to any member of the set ofmolecules that can be joined together to form an oligomer or polymer.The set of monomers useful in the present invention includes, but is notrestricted to, for the example of (poly)peptide synthesis, the set ofL-amino acids, D-amino acids, or synthetic amino acids. As used herein,“monomer” refers to any member of a basis set for synthesis of anoligomer. For example, dimers of L-amino acids form a basis set of 400“monomers” for synthesis of polypeptides. Different basis sets ofmonomers may be used at successive steps in the synthesis of a polymer.The term “monomer” also refers to a chemical subunit that can becombined with a different chemical subunit to form a compound largerthan either subunit alone.

The term “mRNA” or “mRNA transcripts” as used herein, include, but notlimited to pre-mRNA transcript(s), transcript processing intermediates,mature mRNA(s) ready for translation and transcripts of the gene orgenes, or nucleic acids derived from the mRNA transcript(s). Transcriptprocessing may include splicing, editing and degradation. As usedherein, a nucleic acid derived from an mRNA transcript refers to anucleic acid for whose synthesis the mRNA transcript or a subsequencethereof has ultimately served as a template. Thus, a cDNA reversetranscribed from an mRNA, an RNA transcribed from that cDNA, a DNAamplified from the cDNA, an RNA transcribed from the amplified DNA,etc., are all derived from the mRNA transcript and detection of suchderived products is indicative of the presence and/or abundance of theoriginal transcript in a sample. Thus, mRNA derived samples include, butare not limited to, mRNA transcripts of the gene or genes, cDNA reversetranscribed from the mRNA, cRNA transcribed from the cDNA, DNA amplifiedfrom the genes, RNA transcribed from amplified DNA, and the like.

The term “nucleic acid library or array” as used herein refers to anintentionally created collection of nucleic acids which can be preparedeither synthetically or biosynthetically and screened for biologicalactivity in a variety of different formats (e.g., libraries of solublemolecules; and libraries of oligos tethered to resin beads, silicachips, or other solid supports). Additionally, the term “array” is meantto include those libraries of nucleic acids which can be prepared byspotting nucleic acids of essentially any length (e.g., from 1 to about1000 nucleotide monomers in length) onto a substrate. The term “nucleicacid” as used herein refers to a polymeric form of nucleotides of anylength, either ribonucleotides, deoxyribonucleotides or peptide nucleicacids (PNAs), that comprise purine and pyrimidine bases, or othernatural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. The backbone of the polynucleotide cancomprise sugars and phosphate groups, as may typically be found in RNAor DNA, or modified or substituted sugar or phosphate groups. Apolynucleotide may comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs. The sequence of nucleotides may beinterrupted by non-nucleotide components. Thus the terms nucleoside,nucleotide, deoxynucleoside and deoxynucleotide generally includeanalogs such as those described herein. These analogs are thosemolecules having some structural features in common with a naturallyoccurring nucleoside or nucleotide such that when incorporated into anucleic acid or oligonucleoside sequence, they allow hybridization witha naturally occurring nucleic acid sequence in solution. Typically,these analogs are derived from naturally occurring nucleosides andnucleotides by replacing and/or modifying the base, the ribose or thephosphodiester moiety. The changes can be tailor made to stabilize ordestabilize hybrid formation or enhance the specificity of hybridizationwith a complementary nucleic acid sequence as desired.

The term “nucleic acids” as used herein may include any polymer oroligomer of pyrimidine and purine bases, preferably cytosine, thymine,and uracil, and adenine and guanine, respectively. See Albert L.Lehninger, PRINCIPLES OF BIOCHEMISTRY, at 793-800 (Worth Pub. 1982).Indeed, the present invention contemplates any deoxyribonucleotide,ribonucleotide or peptide nucleic acid component, and any chemicalvariants thereof, such as methylated, hydroxymethylated or glucosylatedforms of these bases, and the like. The polymers or oligomers may beheterogeneous or homogeneous in composition, and may be isolated fromnaturally-occurring sources or may be artificially or syntheticallyproduced. In addition, the nucleic acids may be DNA or RNA, or a mixturethereof, and may exist permanently or transitionally in single-strandedor double-stranded form, including homoduplex, heteroduplex, and hybridstates.

The term “oligonucleotide” or “polynucleotide” as used herein refers toa nucleic acid ranging from at least 2, preferable at least 8, and morepreferably at least 20 nucleotides in length or a compound thatspecifically hybridizes to a polynucleotide. Polynucleotides of thepresent invention include sequences of deoxyribonucleic acid (DNA) orribonucleic acid (RNA) which may be isolated from natural sources,recombinantly produced or artificially synthesized and mimetics thereof.A further example of a polynucleotide of the present invention may bepeptide nucleic acid (PNA). The invention also encompasses situations inwhich there is a nontraditional base pairing such as Hoogsteen basepairing which has been identified in certain tRNA molecules andpostulated to exist in a triple helix. “Polynucleotide” and“oligonucleotide” are used interchangeably in this application.

The term “probe” as used herein refers to a surface-immobilized moleculethat can be recognized by a particular target. See U.S. Pat. No.6,582,908 for an example of arrays having all possible combinations ofprobes with 10, 12, and more bases. Examples of probes that can beinvestigated by this invention include, but are not restricted to,agonists and antagonists for cell membrane receptors, toxins and venoms,viral epitopes, hormones (e.g., opioid peptides, steroids, etc.),hormone receptors, peptides, enzymes, enzyme substrates, cofactors,drugs, lectins, sugars, oligonucleotides, nucleic acids,oligosaccharides, proteins, and monoclonal antibodies.

The term “primer” as used herein refers to a single-strandedoligonucleotide capable of acting as a point of initiation fortemplate-directed DNA synthesis under suitable conditions e.g., bufferand temperature, in the presence of four different nucleosidetriphosphates and an agent for polymerization, such as, for example, DNAor RNA polymerase or reverse transcriptase. The length of the primer, inany given case, depends on, for example, the intended use of the primer,and generally ranges from 15 to 30 nucleotides. Short primer moleculesgenerally require cooler temperatures to form sufficiently stable hybridcomplexes with the template. A primer need not reflect the exactsequence of the template but must be sufficiently complementary tohybridize with such template. The primer site is the area of thetemplate to which a primer hybridizes. The primer pair is a set ofprimers including a 5′ upstream primer that hybridizes with the 5′ endof the sequence to be amplified and a 3′ downstream primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

The term “polymorphism” as used herein refers to the occurrence of twoor more genetically determined alternative sequences or alleles in apopulation. A polymorphic marker or site is the locus at whichdivergence occurs. Preferred markers have at least two alleles, eachoccurring at frequency of greater than 1%, and more preferably greaterthan 10% or 20% of a selected population. A polymorphism may compriseone or more base changes, an insertion, a repeat, or a deletion. Apolymorphic locus may be as small as one base pair. Polymorphic markersinclude restriction fragment length polymorphisms, variable number oftandem repeats (VNTR's), hypervariable regions, minisatellites,dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats,simple sequence repeats, and insertion elements such as Alu. The firstidentified allelic form is arbitrarily designated as the reference formand other allelic forms are designated as alternative or variantalleles. The allelic form occurring most frequently in a selectedpopulation is sometimes referred to as the wildtype form. Diploidorganisms may be homozygous or heterozygous for allelic forms. Adiallelic polymorphism has two forms. A triallelic polymorphism hasthree forms. Single nucleotide polymorphisms (SNPs) are included inpolymorphisms.

The term “receptor” as used herein refers to a molecule that has anaffinity for a given ligand. Receptors may be naturally-occurring ormanmade molecules. Also, they can be employed in their unaltered stateor as aggregates with other species. Receptors may be attached,covalently or noncovalently, to a binding member, either directly or viaa specific binding substance. Examples of receptors which can beemployed by this invention include, but are not restricted to,antibodies, cell membrane receptors, monoclonal antibodies and antiserareactive with specific antigenic determinants (such as on viruses, cellsor other materials), drugs, polynucleotides, nucleic acids, peptides,cofactors, lectins, sugars, polysaccharides, cells, cellular membranes,and organelles. Receptors are sometimes referred to in the art asanti-ligands. As the term receptors is used herein, no difference inmeaning is intended. A “Ligand Receptor Pair” is formed when twomacromolecules have combined through molecular recognition to form acomplex. Other examples of receptors which can be investigated by thisinvention include but are not restricted to those molecules shown inU.S. Pat. No. 5,143,854, which is hereby incorporated by reference inits entirety.

The term “solid support”, “support”, and “substrate” as used herein areused interchangeably and refer to a material or group of materialshaving a rigid or semi-rigid surface or surfaces. In many embodiments,at least one surface of the solid support will be substantially flat,although in some embodiments it may be desirable to physically separatesynthesis regions for different compounds with, for example, wells,raised regions, pins, etched trenches, or the like. According to otherembodiments, the solid support(s) will take the form of beads, resins,gels, microspheres, or other geometric configurations. See U.S. Pat. No.5,744,305 for exemplary substrates.

The term “target” as used herein refers to a molecule that has anaffinity for a given probe. Targets may be naturally-occurring orman-made molecules. Also, they can be employed in their unaltered stateor as aggregates with other species. Targets may be attached, covalentlyor noncovalently, to a binding member, either directly or via a specificbinding substance. Examples of targets which can be employed by thisinvention include, but are not restricted to, antibodies, cell membranereceptors, monoclonal antibodies and antisera reactive with specificantigenic determinants (such as on viruses, cells or other materials),drugs, oligonucleotides, nucleic acids, peptides, cofactors, lectins,sugars, polysaccharides, cells, cellular membranes, and organelles.Targets are sometimes referred to in the art as anti-probes. As the termtargets is used herein, no difference in meaning is intended. A “ProbeTarget Pair” is formed when two macromolecules have combined throughmolecular recognition to form a complex.

c) EMBODIMENTS OF THE PRESENT INVENTION

Embodiments of an imaging system, methods, and product are describedthat generates an interference pattern employed for embodiments ofdetection schemes that resolve very small feature sizes. For example,some embodiments of probe arrays include very small probe features thatmay include square, rectangular, hexagonal, round, or other shape ofprobe feature with a maximum dimension that is no greater than 1 μm(i.e. largest dimension such as a side or radius or more generallydistance between any two points of a probe feature is 1 μm or less). Inthe present example, probe features of small dimension are difficult toresolve using standard methods and instrumentation. In response,detections schemes that may be referred to as patterned illumination orpatterned excitation have been developed as a solution to theseresolution problems. A key element to the described detection schemes isthe generation of an interference pattern that can be controlled forcharacteristics such as phase, and orientation. The presently describedinvention provides a simple and efficient means for generating suchinterference patterns.

Probe Array 140: An illustrative example of probe array 140 is providedin FIGS. 1, and 3. Descriptions of probe arrays are provided above withrespect to “Nucleic Acid Probe arrays” and other related disclosure. Invarious implementations, probe array 140 may be disposed in a cartridgeor housing such as, for example, the GeneChip® probe array availablefrom Affymetrix, Inc. of Santa Clara Calif. Examples of probe arrays andassociated cartridges or housings may be found in U.S. Pat. Nos.5,945,334, 6,287,850, 6,399,365, 6,551,817, each of which is also herebyincorporated by reference herein in its entirety for all purposes. Inaddition, some embodiments of probe array 140 may be associated withpegs or posts. For instance probe array 140 may be affixed to a peg orpost via gluing, welding, or other means known in the related art. Alsothe peg or post may be operatively coupled to a tray, strip, or othertype of similar structure, where probe array 140 is extended away fromthe tray, strip, or structure by a distance measured by the height ofthe peg or post. Examples with embodiments of probe array 140 associatedwith pegs or posts may be found in U.S. patent Ser. No. 10/826,577,titled “Immersion Array Plates for Interchangeable Microtiter WellPlates”, filed Apr. 16, 2004, which is hereby incorporated by referenceherein in its entirety for all purposes.

Scanner 100: Labeled targets hybridized to probe arrays may be detectedusing various devices, sometimes referred to as scanners, as describedabove with respect to methods and apparatus for signal detection. Anillustrative device is shown in FIG. 1 as scanner 100. For example, someembodiments of scanners image targets by detecting fluorescent or otheremissions from labels associated with target molecules, or by detectingtransmitted, reflected, or scattered radiation. A typical scheme employsoptical and other elements to provide excitation light and toselectively collect the emissions.

For example, scanner 100 provides a signal representing the intensities(and possibly other characteristics, such as color that may beassociated with a detected wavelength) of the detected emissions orreflected wavelengths of light, as well as the locations on thesubstrate where the emissions or reflected wavelengths were detected.Typically, the signal includes intensity information corresponding toelemental sub-areas of the scanned substrate. The term “elemental” inthis context means that the intensities, and/or other characteristics,of the emissions or reflected wavelengths from this area each arerepresented by a single value. When displayed as an image for viewing orprocessing, elemental picture elements, or pixels, often represent thisinformation. Thus, in the present example, a pixel may have a singlevalue representing the intensity of the elemental sub-area of thesubstrate from which the emissions or reflected wavelengths werescanned. The pixel may also have another value representing anothercharacteristic, such as color, positive or negative image, or other typeof image representation. The size of a pixel may vary in differentembodiments and could include a 2.5 μm, 1.5 μm, 1.0 μm, or sub-micronpixel size. Examples where the signal may be incorporated into datafiles may include incorporating data according to the well known *.dator *.tif file formats as generated respectively by instrument controland image processing applications 372 (described in greater detailbelow).

Embodiments of scanner 100 may include various elements and/or opticalarchitectures enabled for fluorescent detection. For instance, someembodiments of scanner 100 may employ what is referred to as a“confocal” type architecture that may include the use of photomultipliertubes to as detection elements. Alternatively, some embodiments ofscanner 100 may employ a CCD type (referred to as a Charge CoupledDevice) architecture using what is referred to as a CCD or cooled CCDcameras as detection elements. Further examples of scanner systems thatmay be implemented with embodiments of the present invention includeU.S. patent application Ser. No. 10/389,194, 10/846,261, 10/913,102, and11/260,617; each of which are incorporated by reference above; and U.S.patent application Ser. No. 11/379,641, titled “Methods and Devices forReading Microarrays”, filed Apr. 21, 2006, which is hereby incorporatedby reference herein in its entirety for all purposes.

As described above, some embodiments of scanner 100 may be enabled toemploy diction schemes referred to as “Patterned Excitation” or“Patterned Illumination”. A key element of this type of detection schemeis the generation and manipulation of an interference pattern, typicallycomprising excitation light in a pattern where characteristics such asthe phase and orientation are controlled. For example, scanner 100 mayinclude an array of mirrors and a source of excitation light that mayinclude a solid state laser that emits a wavelength of 532 nm. Scanner100 may also include a beam splitter that directs a portion of the lightemitted from the laser into a separate optical path, thus creating twooptical paths where the degree of power associated with the light ineach path is substantially the same. In some embodiments, lighttraveling along one path will be reflected by a mirror that may betranslated to increase or decrease the distance of travel by the lightbeam. Those of ordinary skill in the related art will appreciate thatthe phase of the light in the two paths may be altered relative to oneanother by manipulating the distance traveled by one path relative tothe other. In the present example, scanner 100 may cause the two beamsto converge at various angles to produce interference patterns indesired orientations and phases. Examples of systems and methods thatemploy such detection schemes are described in U.S. patent applicationSer. Nos. 11/026,615, titled “Methods and Devices for Microarray ImageAnalysis”, filed Dec. 30, 2004; 11/101,019, titled “Methods and Devicesfor Microarray Image Analysis”, filed Apr. 6, 2005; 11/101,043, titled“Methods and Devices for Microarray Image Analysis”, filed Apr. 6, 2005;and 11/101,087, titled “Methods and Devices for Microarray ImageAnalysis”, filed Apr. 6, 2005, each of which is hereby incorporated byreference herein in its entirety for all purposes.

Computer 150: An illustrative example of computer 150 is provided inFIG. 1 and also in greater detail in FIG. 2. Computer 150 may be anytype of computer platform such as a workstation, a personal computer, aserver, or any other present or future computer. Computer 150 typicallyincludes known components such as a processor 255, an operating system260, system memory 270, memory storage devices 281, and input-outputcontrollers 275, input-output devices 240, and display devices 245.Display devices 245 may include display devices that provides visualinformation, this information typically may be logically and/orphysically organized as an array of pixels. An interface controller mayalso be included that may comprise any of a variety of known or futuresoftware programs for providing input and output interfaces such as forinstance interfaces 246. For example, interfaces 246 may include whatare generally referred to as “Graphical User Interfaces” (often referredto as GUI's) that provide one or more graphical representations to auser, such as user 101. Interfaces 246 are typically enabled to acceptuser inputs using means of selection or input known to those of ordinaryskill in the related art.

In the same or alternative embodiments, applications on computer 150 mayemploy interface 246 that include what are referred to as “command lineinterfaces” (often referred to as CLI's). CLI's typically provide a textbased interaction between the application and user 101. Typically,command line interfaces present output and receive input as lines oftext through display devices 245. For example, some implementations mayinclude what are referred to as a “shell” such as Unix Shells known tothose of ordinary skill in the related art, or Microsoft WindowsPowershell that employs object-oriented type programming architecturessuch as the Microsoft.NET framework.

Those of ordinary skill in the related art will appreciate thatinterfaces 246 may include one or more GUI's, CLI's or a combinationthereof.

It will be understood by those of ordinary skill in the relevant artthat there are many possible configurations of the components ofcomputer 150 and that some components that may typically be included incomputer 150 are not shown, such as cache memory, a data backup unit,and many other devices. Processor 255 may be a commercially availableprocessor such as an Itanium® or Pentium® processor made by IntelCorporation, a SPARC® processor made by Sun Microsystems, an Athalon™ orOpteron™ processor made by AMD corporation, or it may be one of otherprocessors that are or will become available. Some embodiments ofprocessor 255 may also include what are referred to as Multi-coreprocessors and/or be enabled to employ parallel processing technology ina single or multi-core configuration. For example, a multi-corearchitecture typically comprises two or more processor “executioncores”. In the present example each execution core may perform as anindependent processor that enables parallel execution of multiplethreads. In addition, those of ordinary skill in the related willappreciate that processor 255 may be configured in what is generallyreferred to as 32 or 64 bit architectures, or other architecturalconfigurations now known or that may be developed in the future.

Processor 255 executes operating system 260, which may be, for example,a Windows®-type operating system (such as Windows® XP) from theMicrosoft Corporation; the Mac OS X operating system from Apple ComputerCorp. (such as 7.5 Mac OS X v10.4 “Tiger” or 7.6 Mac OS X v10.5“Leopard” operating systems); a Unix® or Linux-type operating systemavailable from many vendors or what is referred to as an open source;another or a future operating system; or some combination thereof.Operating system 260 interfaces with firmware and hardware in awell-known manner, and facilitates processor 255 in coordinating andexecuting the functions of various computer programs that may be writtenin a variety of programming languages. Operating system 260, typicallyin cooperation with processor 255, coordinates and executes functions ofthe other components of computer 150. Operating system 260 also providesscheduling, input-output control, file and data management, memorymanagement, and communication control and related services, all inaccordance with known techniques.

System memory 270 may be any of a variety of known or future memorystorage devices. Examples include any commonly available random accessmemory (RAM), magnetic medium such as a resident hard disk or tape, anoptical medium such as a read and write compact disc, or other memorystorage device. Memory storage devices 281 may be any of a variety ofknown or future devices, including a compact disk drive, a tape drive, aremovable hard disk drive, USB or flash drive, or a diskette drive. Suchtypes of memory storage devices 281 typically read from, and/or writeto, a program storage medium (not shown) such as, respectively, acompact disk, magnetic tape, removable hard disk, USB or flash drive, orfloppy diskette. Any of these program storage media, or others now inuse or that may later be developed, may be considered a computer programproduct. As will be appreciated, these program storage media typicallystore a computer software program and/or data. Computer softwareprograms, also called computer control logic, typically are stored insystem memory 270 and/or the program storage device used in conjunctionwith memory storage device 281.

In some embodiments, a computer program product is described comprisinga computer usable medium having control logic (computer softwareprogram, including program code) stored therein. The control logic, whenexecuted by processor 255, causes processor 255 to perform functionsdescribed herein. In other embodiments, some functions are implementedprimarily in hardware using, for example, a hardware state machine.Implementation of the hardware state machine so as to perform thefunctions described herein will be apparent to those skilled in therelevant arts.

Input-output controllers 275 could include any of a variety of knowndevices for accepting and processing information from a user, whether ahuman or a machine, whether local or remote. Such devices include, forexample, modem cards, wireless cards, network interface cards, soundcards, or other types of controllers for any of a variety of known inputdevices. Output controllers of input-output controllers 275 couldinclude controllers for any of a variety of known display devices forpresenting information to a user, whether a human or a machine, whetherlocal or remote. In the illustrated embodiment, the functional elementsof computer 150 communicate with each other via system bus 290. Some ofthese communications may be accomplished in alternative embodimentsusing network or other types of remote communications.

As will be evident to those skilled in the relevant art, an instrumentcontrol and image processing application, such as for instance animplementation of instrument control and image processing applications372 illustrated in FIG. 3, if implemented in software, may be loadedinto and executed from system memory 270 and/or memory storage device281. All or portions of the instrument control and image processingapplications may also reside in a read-only memory or similar device ofmemory storage device 281, such devices not requiring that theinstrument control and image processing applications first be loadedthrough input-output controllers 275. It will be understood by thoseskilled in the relevant art that the instrument control and imageprocessing applications 372, or portions of it, may be loaded byprocessor 255 in a known manner into system memory 270, or cache memory(not shown), or both, as advantageous for execution. Also illustrated inFIG. 2 are library files 274, experiment data 277, and internet client279 stored in system memory 270. For example, experiment data 277 couldinclude data related to one or more experiments or assays such asexcitation wavelength ranges, emission wavelength ranges, extinctioncoefficients and/or associated excitation power level values, or othervalues associated with one or more fluorescent labels. Additionally,internet client 279 may include an application enabled to accesses aremote service on another computer using a network that may for instancecomprise what are generally referred to as “Web Browsers”. In thepresent example some commonly employed web browsers include Netscape®8.0 available from Netscape Communications Corp., Microsoft® InternetExplorer 6 with SP1 available from Microsoft Corporation, MozillaFirefox® 1.5 from the Mozilla Corporation, Safari 2.0 from AppleComputer Corp., or other type of web browser currently known in the artor to be developed in the future. Also, in the same or other embodimentsinternet client 279 may include, or could be an element of, specializedsoftware applications enabled to access remote information via a networksuch as network 125 such as, for instance, the GeneChip® Data AnalysisSoftware (GDAS) package or Chromosome Copy Number Tool (CNAT) bothavailable from Affymetrix, Inc. of Santa Clara Calif. that are eachenabled to access information from remote sources, and in particularprobe array annotation information from the NetAffx™ web site hosted onone or more servers provided by Affymetrix, Inc.

Network 125 may include one or more of the many various types ofnetworks well known to those of ordinary skill in the art. For example,network 125 may include a local or wide area network that employs whatis commonly referred to as a TCP/IP protocol suite to communicate.Network 125 may include a network comprising a worldwide system ofinterconnected computer networks that is commonly referred to as theinternet, or could also include various intranet architectures. Those ofordinary skill in the related arts will also appreciate that some usersin networked environments may prefer to employ what are generallyreferred to as “firewalls” (also sometimes referred to as PacketFilters, or Border Protection Devices) to control information traffic toand from hardware and/or software systems. For example, firewalls maycomprise hardware or software elements or some combination thereof andare typically designed to enforce security policies put in place byusers, such as for instance network administrators, etc.

Server 120: FIG. 1 shows a typical configuration of a server computerconnected to a workstation computer via a network that is illustrated infurther detail in FIG. 3. In some implementations any function ascribedto server 120 may be carried out by one or more other computers, and/orthe functions may be performed in parallel by a group of computers.

Typically, server 120 is a network-server class of computer designed forservicing a number of workstations or other computer platforms over anetwork. However, server 120 may be any of a variety of types ofgeneral-purpose computers such as a personal computer, workstation, mainframe computer, or other computer platform now or later developed.Server 120 typically includes known components such as processor 355,operating system 360, system memory 370, memory storage devices 381, andinput-output controllers 375. It will be understood by those skilled inthe relevant art that there are many possible configurations of thecomponents of server 120 that may typically include cache memory, a databackup unit, and many other devices. Similarly, many hardware andassociated software or firmware components may be implemented in anetwork server. For example, components to implement one or morefirewalls to protect data and applications, uninterruptible powersupplies, LAN switches, web-server routing software, and many othercomponents. Those of ordinary skill in the art will readily appreciatehow these and other conventional components may be implemented.

Processor 355 may include multiple processors; e.g., multiple Intel®Xeon™ 3.2 GHz processors. As further examples, the processor may includeone or more of a variety of other commercially available processors suchas Itanium® 2 64-bit processors or Pentium® processors from Intel,SPARC® processors made by Sun Microsystems, Opteron™ processors fromAdvanced Micro Devices, or other processors that are or will becomeavailable. Processor 355 executes operating system 360, which may be,for example, a Windows®-type operating system (such as Windows® XPProfessional (which may include a version of Internet Information Server(IIS))) from the Microsoft Corporation; the Mac OS X Server operatingsystem from Apple Computer Corp.; the Solaris operating system from SunMicrosystems; the Tru64 Unix from Compaq; other Unix® or Linux-typeoperating systems available from many vendors or open sources; anotheror a future operating system; or some combination thereof. Someembodiments of processor 355 may also include what are referred to asMulti-core processors and/or be enabled to employ parallel processingtechnology in a single or multi-core configuration similar to that asdescribed above with respect to processor 255. In addition, those ofordinary skill in the related will appreciate that processor 355 may beconfigured in what is generally referred to as 32 or 64 bitarchitectures, or other architectural configurations now known or thatmay be developed in the future.

Operating system 360 interfaces with firmware and hardware in awell-known manner, and facilitates processor 355 in coordinating andexecuting the functions of various computer programs that may be writtenin a variety of programming languages. Operating system 360, typicallyin cooperation with the processor, coordinates and executes functions ofthe other components of server 120. Operating system 360 also providesscheduling, input-output control, file and data management, memorymanagement, and communication control and related services, all inaccordance with known techniques.

System memory 370 may be any of a variety of known or future memorystorage devices. Examples include any commonly available random accessmemory (RAM), magnetic medium such as a resident hard disk or tape, anoptical medium such as a read and write compact disc, or other memorystorage device. Memory storage device 381 may be any of a variety ofknown or future devices, including a compact disk drive, a tape drive, aremovable hard disk drive, USB or flash drive, or a diskette drive. Suchtypes of memory storage device typically read from, and/or write to, aprogram storage medium (not shown) such as, respectively, a compactdisk, magnetic tape, removable hard disk, USB or flash drive, or floppydiskette. Any of these program storage media, or others now in use orthat may later be developed, may be considered a computer programproduct. As will be appreciated, these program storage media typicallystore a computer software program and/or data. Computer softwareprograms, also called computer control logic, typically are stored inthe system memory and/or the program storage device used in conjunctionwith the memory storage device.

In some embodiments, a computer program product is described comprisinga computer usable medium having control logic (computer softwareprogram, including program code) stored therein. The control logic, whenexecuted by the processor, causes the processor to perform functionsdescribed herein. In other embodiments, some functions are implementedprimarily in hardware using, for example, a hardware state machine.Implementation of the hardware state machine so as to perform thefunctions described herein will be apparent to those skilled in therelevant arts.

Input-output controllers 375 could include any of a variety of knowndevices for accepting and processing information from a user, whether ahuman or a machine, whether local or remote. Such devices include, forexample, modem cards, network interface cards, sound cards, or othertypes of controllers for any of a variety of known input or outputdevices. In the illustrated embodiment, the functional elements ofserver 120 communicate with each other via system bus 390. Some of thesecommunications may be accomplished in alternative embodiments usingnetwork or other types of remote communications.

As will be evident to those skilled in the relevant art, a serverapplication if implemented in software may be loaded into the systemmemory and/or the memory storage device through one of the inputdevices, such as instrument control and image processing applications372 described in greater detail below. All or portions of these loadedelements may also reside in a read-only memory or similar device of thememory storage device, such devices not requiring that the elementsfirst be loaded through the input devices. It will be understood bythose skilled in the relevant art that any of the loaded elements, orportions of them, may be loaded by the processor in a known manner intothe system memory, or cache memory (not shown), or both, as advantageousfor execution.

Instrument control and image processing applications 372: Instrumentcontrol and image processing applications 372 may comprise any of avariety of known or future image processing applications. Some examplesof known instrument control and image processing applications includethe Affymetrix® Microarray Suite, and Affymetrix® GeneChip® OperatingSoftware (hereafter referred to as GCOS) applications. Typically,embodiments of applications 372 may be loaded into system memory 370and/or memory storage device 381. For example, FIG. 3 provides anexample of applications 372 stored for execution in system memory 370illustrated as instrument control and image processing applicationsexecutables 372A. Also, those of ordinary skill in the related art willappreciate that applications 372 may be stored for execution on anycompatible computer system, such as computer 150. For example, thedescribed embodiments of applications 372 may, for example, include theAffymetrix® Command Console™ software application.

Embodiments of applications 372 may provide what is referred to as amodular interface for one or more computers or workstations and one ormore servers, as well as one or more instruments. The term “modular” asused herein generally refers to elements that may be integrated to andinteract with a core element in order to provide a flexible, updateable,and customizable platform. For example, as will be described in greaterdetail below applications 372 may include a “core” software elementenabled to communicate and perform primary functions necessary for anyinstrument control and image processing application. Such primaryfunctionality may include communication over various networkarchitectures, or data processing functions such as processing rawintensity data into a .dat file. In the present example, modularsoftware elements, such as for instance what may be referred to as aplug-in module, may be interfaced with the core software element toperform more specific or secondary functions, such as for instancefunctions that are specific to particular instruments. In particular,the specific or secondary functions may include functions customizablefor particular applications desired by user 101. Further, integratedmodules and the core software element are considered to be a singlesoftware application, and referred to as applications 372.

In the presently described implementation, applications 372 maycommunicate with, and receive instruction or information from, orcontrol one or more elements or processes of one or more servers, one ormore workstations, and one or more instruments. Also, embodiments ofserver 120 or computer 150 with an implementation of applications 372stored thereon could be located locally or remotely and communicate withone or more additional servers and/or one or more othercomputers/workstations or instruments.

In some embodiments, applications 372 may be capable of dataencryption/decryption functionality. For example, it may be desirable toencrypt data, files, information associated with GUI's 246, or otherinformation that may be transferred over network 125 to one or moreremote computers or servers for data security and confidentialitypurposes. For example, some embodiments of probe array 140 may beemployed for diagnostic purposes where the data may be associated with apatient and/or a diagnosis of a disease or medical condition. It isdesirable in many applications to protect the data using encryption forconfidentiality of patient information. In addition, one-way encryptiontechnologies may be employed in situations where access should belimited to only selected parties such as a patient and their physician.In the present example, only the selected parties have the key todecrypt or associate the data with the patient. In some applications,the one-way encrypted data may be stored in one or more public databasesor repositories where even the curator of the database or repositorywould be unable to associate the data with the user or otherwise decryptthe information. The described encryption functionality may also haveutility in clinical trial applications where it may be desirable toisolate one or more data elements from each other for the purpose ofconfidentiality and/or removal of experimental biases.

Various embodiments of applications 372 may provide one or moreembodiments of interfaces 246 that may include interactive graphicaluser interfaces that allows user 101 to make selections based uponinformation presented in an embodiment of interface 246. Those ofordinary skill will recognize that embodiments of interface 246 mayinclude GUI's as described above coded in various language formats suchas an HTML, XHTML, XML, javascript, Jscript, or other language known tothose of ordinary skill in the art used for the creation or enhancementof “Web Pages” viewable and compatible with internet client 279 or 379.For example, internet client 279 or 379 may include various internetbrowsers such as Microsoft Internet Explorer, Netscape Navigator,Mozilla Firefox, Apple Safari, or other browsers known in the art.Applications of GUI's viewable via one or more browsers may allow user101 complete remote access to data, management, and registrationfunctions without any other specialized software elements. Applications372 may provide one or more implementations of interactive GUI's thatallow user 101 to select from a variety of options including dataselection, experiment parameters, calibration values, and probe arrayinformation within the access to data, management, and registrationfunctions.

In some embodiments, applications 372 may be capable of running onoperating systems in a non-English format, where applications 372 canaccept input from user 101 via interface 246 in various non-Englishlanguage formats such as Chinese, French, Spanish etc., and outputinformation to user 101 in the same or other desired language output.For example, applications 372 may present information to user 101 invarious implementations of a GUI in a language output desired by user101, and similarly receive input from user 101 in the desired language.In the present example, applications 372 is internationalized such thatit is capable of interpreting the input from user 101 in the desiredlanguage where the input is acceptable input with respect to thefunctions and capabilities of applications 372.

Embodiments of applications 372 also include instrument controlfeatures, where the control functions of individual types or specificinstruments such as scanner 100, an autoloader, or a fluid processingsystem may be organized as plug-in type modules 373 to applications 372.For example, each plug-in module 373 may be a separate component and mayprovide definition of the instrument control features to applications372. As described above, each plug-in module 373 is functionallyintegrated with applications 372 when stored in system memory 270 andthus reference to applications 372 includes any embodiments ofintegrated plug-in modules 373. In the present example, each instrumentmay have one or more associated embodiments of plug-in module 373 thatfor instance may be specific to model of instrument, revision ofinstrument firmware or scripts, number and/or configuration ofinstrument embodiment, etc. Further, multiple embodiments of plug-inmodule 373 for the same instrument such as scanner 100 may be stored insystem memory 270 for use by applications 372, where user 101 may selectthe desired embodiment of module to employ, or alternatively such aselection of module may be defined by data encoded directly in a machinereadable identifier or indirectly via the array file, library files,experiments files and so on.

The instrument control features may include the control of one or moreelements of one or more instruments that could, for instance, includeelements of a hybridization device, a fluid processing instrument, anautoloader, or scanner 100. The instrument control features may also becapable of receiving information from the one or more instruments thatcould include experiment or instrument status, process steps, or otherrelevant information. The instrument control features could, forexample, be under the control of or an element of the interface ofapplications 372. In some embodiments, a user may input desired controlcommands and/or receive the instrument control information via one ofinterfaces 246. Additional examples of instrument control via a GUI orother interface is provided in U.S. patent application Ser. No.10/764,663, titled “System, Method and Computer Software Product forInstrument Control, Data Acquisition, Analysis, Management and Storage”,filed Jan. 26, 2004, which is hereby incorporated by reference herein inits entirety for all purposes.

In some embodiments, applications 372 may employ what may referred to asan “array file” that comprises data employed for various instruments,processing functions of images by applications 372, or other relevantinformation. Generally it is desirable to consolidate elements of dataor metadata related to an embodiment of probe array 140, experiment,user, or some combination thereof, to a single file that is notduplicated (i.e. as embodiments of .dat file may be in certainapplications), where duplication may sometimes be a source of error. Theterm “metadata” as used herein generally refers to data about data. Itmay also be desirable in some embodiments to restrict or prohibit theability to overwrite data in the array file. Preferentially, newinformation may be appended to the array file rather than deleting oroverwriting information, providing the benefit of traceability and dataintegrity (i.e. as may be required by some regulatory agencies). Forexample, an array file may be associated with one or moreimplementations of an embodiment of probe array 140, where the arrayfile acts to unify data across a set of probe arrays 140. The array filemay be created by applications 372 via a registration process, whereuser 101 inputs data into applications 372 via one or more of interfaces246. In the present example, the array file may be associated by user101 with a custom identifier that could include a machine readableidentifier such as the machine readable identifiers described in greaterdetail below.

Alternatively, applications 372 may create an array file andautomatically associate the array file with a machine readableidentifier that identifies an embodiment of probe array 140 (i.e.relationship between the machine readable identifier and probe array 140may be assigned by a manufacturer). Applications 372 may employ variousdata elements for the creation or update of the array file from one ormore library files, such as library files 274 or other library files.

Also in the same or alternative embodiments, the array file may includepointers to one or more additional data files comprising data related toan associated embodiment of probe array 140. For example, themanufacturer of probe array 140 or other user may provide library files274 or other files that define characteristics such as probe identity;dimension and positional location (i.e. with respect to some fiducialreference or coordinate system) of the active area of probe array 140;various experimental parameters; instrument control parameters; or othertypes of useful information. In addition, the array file may alsocontain one or more metadata elements that could include one or more ofa unique identifier for the array file, human readable form of a machinereadable identifier, or other metadata elements. In addition,applications 372 may store data (i.e. as metadata, or stored data) thatincludes sample identifiers, array names, user parameters, event logsthat may for instance include a value identifying the number of times anarray has been scanned, relationship histories such as for instance therelationship between each .cel file and the one or more .dat files thatwere employed to generate the .cel file, and other types of data usefulin for processing and data management.

For example, user 101 and/or automated data input devices or programs(not shown) may provide data related to the design or conduct ofexperiments. User 101 may specify an Affymetrix catalogue or custom chiptype (e.g., Human Genome U133 plus 2.0 chip) either by selecting from apredetermined list presented in one or more of interfaces 246 or byscanning a bar code, Radio Frequency Identification (RFID), magneticstrip, or other means of electronic identification related to probearray 140 to read its type, part no., array identifier, etc.Applications 372 may associate the chip type, part no., array identifierwith various scanning parameters stored in data tables or library files,such as library files 274 of computer 150, including the area of probearray 140 that is to be scanned, the location of chrome elements orother features on probe array 140 used for auto-focusing, the wavelengthor intensity/power of excitation light to be used in reading the chip,and so on. Also, some embodiments of applications 372 may encode arrayfiles in a binary type format that may minimize the possibility of datacorruption. However, applications 372 may be further enabled to exportan array file in a number of different formats.

Also continuing the example above, some embodiments of RFID tagsassociated with embodiments of probe array 140 may be capable of “datalogging” functionality where, for instance, each RFID tag or label mayactively measure and record parameters of interest. In the presentexample, such parameters of interest may include environmentalconditions such as temperature and/or humidity that the implementationof probe array 140 may have been exposed to. In the present example,user 101 may be interested in the environmental conditions because thebiological integrity of some embodiments of probe array 140 may beaffected by exposure to fluctuations of the environment. In someembodiments, applications 372 may extract the recorded environmentalinformation from the RFID tag or label and store it in the array file,or some other file that has a pointer to or from the array file. In thesame or alternative embodiments, applications 372 may monitor theenvironmental conditions exposed to the probe array in real time, whereapplications 372 may regularly monitor information provided by one ormore RFID tags simultaneously. Applications 372 may further analyze andemploy such information for quality control purposes, for datanormalization, or other purposes known in the related art. Some examplesof RFID embodiments capable to recording environmental parametersinclude the ThermAssureRF™ RFID sensor available from Evidencia LLP ofMemphis Tenn., or the Tempsens™ RFID datalogging label available fromExago Pty Ltd. of Australia.

Also, in the same or alternative embodiments, applications 372 maygenerate or access what may be referred to as a “plate” file. The platefile may encode one or more data elements such as pointers to one ormore array files, and preferably may include pointers to a plurality ofarray files.

In some embodiments, raw image data is acquired from scanner 100 andoperated upon by applications 372 to generate intermediate results. Forexample, raw intensity data acquired from scanner 100 may be directed toa .dat file generator and written to data files (*.dat) that comprise anintensity value for each pixel of data acquired from a scan of anembodiment of probe array 140. In the same or alternative embodiments itmay be advantageous to scan sub areas (that may be referred to as subarrays) of probe array 140 where the detected signal for each sub areascanned may be written to an individual embodiment of a .dat file.Continuing with the present example, applications 372 may also encode aunique identifier for each .dat file as well as a pointer to anassociated embodiment of an array file as metadata into each .dat filegenerated. The term “pointer” as used herein generally refers to aprogramming language data type, variable, or data object that referencesanother data object, datatype, variable, etc. using a memory address oridentifier of the referenced element in a memory storage device such asin system memory 270. In some embodiments the pointers comprise theunique identifiers of the files that are the subject of the pointing,such as for instance the pointer in a .dat file comprises the uniqueidentifier of the array file. Additional examples of the generation andimage processing of sub arrays is described in U.S. patent applicationSer. No. 11/289,975, titled “System, Method, and Product for AnalyzingImages Comprising Small Feature Sizes”, filed Nov. 30, 2005, which ishereby incorporated by reference herein in its entirety for all purpose.

Also, applications 372 may also include a .cel file generator that mayproduce one or more .cel files (*.cel) by processing each .dat file.Alternatively, some embodiments of .cel file generator may produce asingle .cel file from processing multiple .dat files such as with theexample of processing multiple sub-arrays described above. Similar tothe .dat file described above each embodiment of .cel file may alsoinclude one or more metadata elements. For example, applications 372 mayencode a unique identifier for each .cel file as well as a pointer to anassociated array file and/or the one or more .dat files used to producethe .cel file.

Each .cel file contains, for each probe feature scanned by scanner 100,a single value representative of the intensities of pixels measured byscanner 100 for that probe feature. For example, this value may includea measure of the abundance of tagged mRNA's present in the sample thathybridized to the corresponding probe molecules disposed in the probefeature. Many such mRNA's may be present in each probe feature, as aprobe feature on a GeneChip® probe array may include, for example,millions of oligonucleotides designed to detect the mRNA's.Alternatively, the value may include a measure related to the sequencecomposition of DNA or other nucleic acid detected by the probes disposedin probe features of a GeneChip® probe array.

As described above, applications 372 receives image data derived fromprobe array 140 using scanner 100 and generates a .dat file that is thenprocessed by applications 372 to produce a .cel intensity file, whereapplications 372 may utilize information from an array file in the imageprocessing function. For instance, a .cel file generator may performwhat is referred to as grid placement methods on the image data in each.dat file using data elements such as dimension information to determineand define the positional location of probe features in the image.Typically, the .cel file generator associates what may be referred to asa grid with the image data in a .dat file for the purpose of determiningthe positional relationship of probe features in the image with theknown positions and identities of the probe features. The accurateregistration of the grid with the image is important for the accuracy ofthe information in the resulting .cel file. Also, some embodiments of.cel file generator may provide user 101 with a graphical representationof a grid aligned to image data from a selected .dat file in animplementation of interface 246 comprising a GUI, and further enableuser 101 to manually refine the position of the grid placement usingmethods commonly employed such as placing a cursor over the grid,selecting such as by holding down a button on a mouse, and dragging thegrid to a preferred positional relationship with the image. Applications372 may then perform methods sometimes referred to as “featureextraction” to assign a value of intensity for each probe represented inthe image as an area defined by the boundary lines of the grid. Examplesof grid registration, methods of positional refinement, and featureextraction are described in U.S. Pat. Nos. 6,090,555; 6,611,767;6,829,376, and U.S. patent application Ser. Nos. 10/391,882, and10/197,369, each of which is hereby incorporated by reference herein inits entirety for all purposes.

As noted, another file that may be generated by applications 372 is a.chp file using a .chp file generator. For example, each .chp file isderived from analysis of a .cel file combined in some cases withinformation derived from an array file, other lab data and/or libraryfiles 274 that specify details regarding the sequences and locations ofprobes and controls. In some embodiments, a machine readable identifierassociated with probe array 140 may indicate the library file directlyor indirectly via one or more identifiers in the array file, to employfor identification of the probes and their positional locations. Theresulting data stored in the .chp file includes degrees ofhybridization, absolute and/or differential (over two or moreexperiments) expression, genotype comparisons, detection ofpolymorphisms and mutations, and other analytical results.

In some alternative embodiments, user 101 may prefer to employ differentapplications to process data such as an independent analysisapplication. An embodiment of an analysis application is illustrated inFIG. 3 as analysis application 380, and also illustrated as stored forexecution in system memory 370 as analysis application executables 380A.Embodiments of analysis application 380 may comprise any of a variety ofknown or probe array analysis applications, and particularly analysisapplications specialized for use with particular embodiments of probearray 140 such as those designed for certain genotyping or expressionapplications. For example, one such embodiment of analysis application380 may include elements that are specialized for analysis of data fromembodiments of probe array 140 comprising probes that interrogate exonregions.

Various embodiments of analysis application 380 may exist such asapplications developed by a probe array manufacturer for specializedembodiments of probe array 140, commercial third party softwareapplications, open source applications, or other applications known inthe art for specific analysis of data from probe arrays 140. Someexamples of known genotyping analysis applications include theAffymetrix® GeneChip® Data Analysis System (GDAS), Affymetrix® GeneChip®Genotyping Analysis Software (GTYPE), Affymetrix® GeneChip® TargetedGenotyping Analysis Software (GTGS), and Affymetrix® GeneChip® SequenceAnalysis Software (GSEQ) applications. Additional examples of genotypinganalysis applications may be found in U.S. patent application Ser. Nos.10/657,481; 10/986,963; and 11/157,768; each of which is herebyincorporated by reference herein in it's entirety for all purposes.Also, some embodiments of analysis application 380 may be employed toimprove data quality or other purposes. An example of such anapplication for removing feature crosstalk is described in U.S. patentapplication Ser. No. 11/427,103, titled “System, Method, and ComputerProduct for Correction of Feature Overlap”, filed Jun. 28, 2006, whichis hereby incorporated by reference herein in its entirety for allpurposes.

Typically, embodiments of analysis applications may be loaded intosystem memory 370 and/or memory storage device 381. For instance, someembodiments of analysis applications 380 include executable code beingstored in system memory 370. Applications 372 may be enabled to export.cel files, .dat files, or other files to an analysis application orenable access to such files on computer 150 by the analysis application.Import and/or export functionality for compatibility with specificsystems or applications may be enabled by one or more integrated modulesas described above with respect to plug-in modules. For example, ananalysis application may be capable of performing specialized analysisof processed intensity data, such as the data in a .cel file. In thepresent example, user 101 may desire to process data associated with aplurality of implementations of probe array 140 and therefore theanalysis application would receive a .cel file associated with eachprobe array for processing. In the present example, applications 372forwards the appropriate files in response to queries or requests fromthe analysis application.

In the same or alternative examples, user 101 and/or the third partydevelopers may employ what are referred to as software development kitsthat enable programmatic access into file formats, or the structure ofapplications 372. Therefore, developers of other software applicationssuch as the described analysis application may integrate with andseamlessly add functionally to or utilize data from applications 372that provides user 101 with a wide range of application and processingcapability. Additional examples of software development kits associatedwith software or data related to probe arrays are described in U.S. Pat.No. 6,954,699, and U.S. application Ser. Nos. 10/764,663 and 11/215,900,each of which is hereby incorporated by reference herein in its entiretyfor all purposes.

Additional examples of .cel and .chp files are described with respect tothe Affymetrix GeneChip® Operating Software or Affymetrix® MicroarraySuite (as described, for example, in U.S. patent application Ser. Nos.10/219,882, and 10/764,663, both of which are hereby incorporated hereinby reference in their entireties for all purposes). For convenience, theterm “file” often is used herein to refer to data generated or used byapplications 372 and executable counterparts of other applications suchas analysis application 380, where the data is written according aformat such as the described .dat, .cel, and .chp formats. Further, thedata files may also be used as input for applications 372 or othersoftware capable of reading the format of the file.

Those of ordinary skill in the related art will appreciate that one ormore operations of applications 372 may be performed by software orfirmware associated with various instruments. For example, scanner 100could include a computer that may include a firmware component thatperforms or controls one or more operations associated with scanner 100.

Yet another example of instrument control and image processingapplications is described in U.S. patent application Ser. No.11/279,068, titled “System, Method and Computer Product for SimplifiedInstrument Control and File Management”, filed Apr. 7, 2006, which ishereby incorporated by reference herein in its entirety for allpurposes.

Interference Pattern Generation: As described above the presentlydescribed invention provides a means for generating an interferencepattern for use in detection schemes employed with embodiments of probearray 140. For example, previously described methods of patterngeneration employ a complex array of mirrors precisely arranged toproduce a number different interference patterns that vary in phase,orientation and other characteristics. The described invention providesan interference pattern that can be varied in orientation and phase aswell, but does not require the complex arrangement of mirrors. Thetypical components for the patterned excitation detection system of theinvention include pattern excitation imaging device and a computersystem for analyzing the images with structured illumination informationand microarray probe feature information.

Some of basic theories and practical devices for generating interferencepatterns on an object to enhance imaging resolution of various objectsare described in, for example, U.S. Provisional Application No.60/559,806, filed on Apr. 6, 2004; and U.S. Provisional Application No.60/565,041, filed on Apr. 23, 2004; and U.S. patent application Ser. No.10/026,615; Jekwan, Ryu, Resolution Improvement in Optical Microscopy byUse of Multibeam Inteferometric Illumination, September 2003, MIT Ph.D.,Dissertation, incorporated herein by reference (including all thereferences cited in the dissertation); J. W. Goodman, Introduction toFourier Optics, McGraw-Hill, Boston, 1996; B. Bailey, D. L. Farkas, D.L. Taylor, F. Lanni, Nature 366, 44 (1993); M. A. A. Neil, R. Juŝkitis,T. Wilson, Opt. Lett 22, 1905 (1997); R. Heintzmann, C. Cremer, Proc.SPIE 3568, 185 (1998); M. G. L. Gustafsson, D. A. Agard, J. W. Sedat, J.Microsc. 195, 10 (1999); M. G. L. Gustafsson, J. Microsc. 198, 82(2000); J. T. Frohn, H. F. Knapp, A. Stemmer, Proc. Natl. Acad. Sci.U.S.A. 97, 7232 (2000); V. Krishnamurthi, B. Bailey, F. Lanni, Proc.SPIE 2655, 18 (1996); G. E. Cragg, P. T. C. So, Opt. Lett 25, 46 (2000);J. T. Frohn, H. F. Knapp, A. Stemmer, Opt. Lett 26, 828 (2001); P. T. C.So, H. S. Kwon, C. Y. Dong, J. Opt. Soc. Am. A 18, 2833 (2001); M. S.Mermelstein, PhD Thesis, Massachusetts Institute of Technology (2000);M. Born, E. Wolf, Principles of Optics (Cambridge University Press,Cambridge, 1980), all incorporated herein by reference.

FIG. 4A provides an illustrative example of a typical “opticalwaveguide” well known in the related art. For instance, opticalwaveguides typically include a physical structure that guideselectromagnetic waves in the optical spectrum. Some examples ofwaveguides include optical fibers or rectangular waveguides and may beclassified by their geometry, mode structure, refractive indexdistribution, and material.

In the example of FIG. 4A, excitation light 405 enters one end of thewaveguide and is directed along optical path 407 and exits through anexit port such as dispersion cone 420 (representing the dispersion oflight that is the effect of diffraction) illuminating an area of asubstrate such as an area associated with probe array 140. Embodimentsof waveguide 410 as illustrated on FIG. 4A may be employed to providewide field illumination to probe array 140.

FIG. 4B provides an illustrative example of an embodiment of twowaveguides positioned adjacent to one another. In the illustratedexample, excitation light 405 enters both waveguide 410A and 410B andtravels along optical path 407A and 407B respectively to exits throughan exit port such as dispersion cones 420A and 420B. As illustrated inFIG. 4B areas of dispersion cone 420A and 420B overlap. As those ofordinary skill will appreciate light acts as waves that diffract(represented by cones 420A and 420B) from each of waveguides 410A and410 B that constructively and destructively interfere with each othercreating an interference pattern as described by what is referred to as“Youngs double slit experiment”.

In the present example, waveguides 410A and 410B may include differentcharacteristics or be manipulated to alter one or more characteristicsof the light traveling in optical paths 407A and 407B. For instance,optical cladding or other similar materials may be applied to one orboth of waveguides 410 to alter the phase relationship between the lightin each path respective to each other. By altering the phaserelationship certain characteristics of the interference pattern may bemanipulated. Also, the waveguide 410A and 410B embodiment may be rotatedabout a central axis to alter the orientation of the interferencepattern.

Also illustrated in FIG. 4B, an interference pattern is generated at asubstrate that may include probe array 140 as described with respect toFIG. 4A. For example, the interference pattern is projected in theregion of dispersion cones 420A and 420B. Further examples ofcommercially available waveguide embodiments for providing interferencepattern include the “Dual Waveguide Interferometer” available fromFarfield Scientific Ltd. of Cheshire, United Kingdom.

An illustrative example of an interference pattern is presented in FIG.5 that includes a regular pattern of alternating bars of illuminationand dark regions. The example of FIG. 5 presents interference pattern505 as a square but those of ordinary skill will appreciate that arepattern 505 may be circular, oblong, or other shape. Therefore thesquare shape of pattern 505 in FIG. 5 should not be interpreted aslimiting.

The interference pattern of area may be controlled for a number ofcharacteristics as previously described and employed for the method ofpatterned excitation/patterned illumination described in U.S. patentapplication Ser. Nos. 11/026,615; 11/101,019; 11/101,043; and11/101,087, incorporated by reference above. Also, those of ordinaryskill in the related art will appreciate that multiple embodiments ofwaveguides 410A and 410B may be employed simultaneously (i.e. eachembodiment may provide an interference pattern with similarcharacteristics) or serially (each embodiment may provide aninterference pattern with different characteristics/orientations).

In one aspect of the invention, a highly sensitive and high speedimaging device, such as an electron multiplying CCD (EM CCD), is used todetect the emission pattern of a hybridized microarray. The microarraycan be a nucleic acid probe array such as a spotted array (e.g., withcDNA or short oligonucleotide probes), high density in situ synthesizedarrays (such as the GeneChip® high density probe arrays manufactured byAffymetrix, Inc., Santa Clara, Calif.). The microarrays can also beprotein or peptide arrays. Typically, the density of the microarrays ishigher than 500, 5000, 50000, or 500,000 different probes per cm². Thefeature size of the probes is typically smaller than 500, 150, 25, 9, or1 μm². The locations of the probes can be determined or decipherable.For example, in some arrays, the specific locations of the probes areknown before binding assays. In some other arrays, the specificlocations of the probes are unknown until after the assays. The probescan be immobilized on a substrate, optionally, via a linker, beads, etc.

An EMCCD device is used for imaging the fluorescence emission pattern,which is used for biological analysis. EM CCD is a device that unitesthe sensitivity of Intensified CCD (ICCD) or an electron bombardment CCD(EBCCD), while retaining the inherent benefits of a CCD. For adescription of the EMCCD technology, see, e.g., EP 08 866 501,incorporated herein by reference. The application of EMCCD enables fastdetection of weak signals. For example, for detecting hybridizationpatterns in nucleic acid probe arrays, the exposure time can be shorterthan 1000, 800, 600, 500, 400, 300, 200, 100, 80, 60, 40, 20, or msec.

In one aspect of the invention, a series of images (frames) of amicroarray, such as a nucleic acid array that has been hybridized with atarget that is labeled with a fluorescent label, are obtained usingpatterned excitation. Such images are then processed based upon theknowledge the excitation patterns employed and the probe featureinformation, such as probe spacing, set backs, feature size, presumeddynamic range, etc.

Microarrays (including bead arrays) typically have periodic repetitionof probes that are synthesized or otherwise immobilized on to asubstrate. The probe features typically assume somewhat regulargeometric shape such as square, rectangular or circular. For example,GeneChip® high density oligonucleotide probe arrays have square featureswith set backs (separation between intended synthesis areas). Theinformation about the periodic repetition of probes is used tofacilitate the extraction of probe intensities from the series of imagesobtained using patterned excitation. Methods for image processing areshown in the patents and applications incorporated by reference above,such as U.S. Ser. No. 11/101,087. U.S. Ser. No. 11/101,087 shows thatPatterned Illumination has been performed on Affymetrix nucleic acidprobe arrays. The present invention modifies that invention to use adifferent illumination source. See also U.S. Ser. No. 11/627,876 formathematical methods to image small feature sizes which is alsoincorporated by reference in its entirety.

One embodiment of the present invention uses a waveguided structuredillumination technique. This technique uses two adjacent waveguides tocreate an interference pattern in order to generate a phase-shift-ableand rotate-able structured illumination pattern for high resolutionimaging for use in microarray imaging and other fields. One preferredembodiment of the invention rotates the waveguide assembly about thebeam axis to alter the angles of the interference pattern with respectto the microarray.

This embodiment of the invention uses a variable index of refractioncladding or similar technology (including labeled biological surface inFarfield's picture) on at least one surface of the optical wave guide totranslate the phase of the interference pattern.

The rotation of the waveguide assembly is significant to this embodimentof the invention. Rotation enables the system to mimic the capability ofa multi mirror system with alternative and potentially advantageoushardware. Repeated fluorescent acquisition could use CCD imagers as oneexample.

An optical waveguide is a physical structure that guides electromagneticwaves in the optical spectrum. Common types of optical waveguidesinclude optical fiber and rectangular waveguides. Optical waveguides areused as components in integrated optical circuits or as the transmissionmedium in local and long haul optical communication systems.

Optical waveguides can be classified according to their geometry(planar, strip, or fiber waveguides), mode structure (single mode, multimode), refractive index distribution (step or gradient index) andmaterial (glass, polymer, semiconductor).

Optical fiber is one example of an optical waveguide and is typically acircular cross-section dielectric waveguide consisting of a dielectricmaterial surrounded by another dielectric material with a lowerrefractive index. Optical fibers are most commonly made from silicaglass, however other glass materials are used for certain applicationsand plastic optical fiber can be used for short-distance applications.Regarding optical fiber in telecommunication, cladding is one or morelayers of material of lower refractive index, in intimate contact with acore material of higher refractive index. Cladding can change the phaseof the excitation light passing through a waveguide.

Having described various embodiments and implementations, it should beapparent to those skilled in the relevant art that the foregoing isillustrative only and not limiting, having been presented by way ofexample only. Many other schemes for distributing functions among thevarious functional elements of the illustrated embodiment are possible.The functions of any element may be carried out in various ways inalternative embodiments.

Also, the functions of several elements may, in alternative embodiments,be carried out by fewer, or a single, element. Similarly, in someembodiments, any functional element may perform fewer, or different,operations than those described with respect to the illustratedembodiment. Also, functional elements presented as distinct for purposesof illustration may be incorporated within other functional elements ina particular implementation. Also, the sequencing of functions orportions of functions generally may be altered. Certain functionalelements, files, data structures, and so on may be described in theillustrated embodiments as located in system memory of a particularcomputer. In other embodiments, however, they may be located on, ordistributed across, computer systems or other platforms that areco-located and/or remote from each other. For example, any one or moreof data files or data structures described as co-located on and “local”to a server or other computer may be located in a computer system orsystems remote from the server. In addition, it will be understood bythose skilled in the relevant art that control and data flows betweenand among functional elements and various data structures may vary inmany ways from the control and data flows described above or indocuments incorporated by reference herein. More particularly,intermediary functional elements may direct control or data flows, andthe functions of various elements may be combined, divided, or otherwiserearranged to allow parallel processing or for other reasons. Also,intermediate data structures or files may be used and various describeddata structures or files may be combined or otherwise arranged. Numerousother embodiments, and modifications thereof, are contemplated asfalling within the scope of the present invention as defined by appendedclaims and equivalents thereto.

1. A method for generating an interference pattern at a probe array,comprising: directing a light at a first waveguide and second waveguide,wherein the light travels along an optical path, wherein the first andsecond waveguides are positioned adjacent to each other and the outputfrom the first and second waveguides produce an orientation of aninterference pattern; rotating at least one waveguide about an axisrelative to the optical path to alter the orientation of theinterference pattern; and directing the interference pattern at theprobe array.
 2. The method according to claim 1, wherein the rotatingstep further comprises rotating the first and second waveguides about acentral axis to alter the orientation.
 3. The method according to claim1, further comprising altering a phase relationship between the light ineach optical path respective to each other, wherein an optical claddingis applied to at least one of the waveguides.
 4. The method according toclaim 1 wherein the probe array has a biopolymer affixed to the surfaceof a support selected from the group consisting of nucleic acids,oligonucleotides, amino acids, proteins, peptides, hormones,oligosaccharides, lipids, glycolipids, lipopolysaccharides,phospholipids, inverted nucleotides, peptide nucleic acids, andMeta-DNA.
 5. The method according to claim 2 wherein the probe array isa nucleic acid or protein probe array.
 6. The method according to claim1 further comprising imaging the probe array using a detector.
 7. Themethod according to claim 6, wherein the detector comprises a CCD, EMCCD, EB CCD, CMOS, APS, or PMT.
 8. The method according to claim 1further comprising processing the detected image using a computersystem.
 9. The method according to claim 1 wherein the waveguides areoptical fibers or rectangular waveguides.
 10. The method for generatingan interference pattern to interrogate a surface of a supportcomprising: directing light at a first waveguide, wherein the lighttravels along a first optical path; directing light at a secondwaveguide, wherein the light travels along a second optical path,wherein the first and second waveguides are positioned in an operativerelationship so as to produce an orientation of an interference pattern;rotating at least one waveguide about an axis relative to the opticalpath to alter the orientation of the interference pattern; directing theinterference pattern at a surface of a support; and detecting signalsfrom the surface of the support.
 11. The method according to claim 10,wherein the rotating step further comprises rotating the first andsecond waveguides about a central axis to alter the orientation.
 12. Themethod according to claim 10, further comprising altering a phaserelationship between the light in each optical path respective to eachother, wherein an optical cladding is applied to at least one of thewaveguides.
 13. The method according to claim 10 wherein the probe arrayhas a biopolymer affixed to the surface of a support selected from thegroup consisting of nucleic acids, oligonucleotides, amino acids,proteins, peptides, hormones, oligosaccharides, lipids, glycolipids,lipopolysaccharides, phospholipids, inverted nucleotides, peptidenucleic acids, and Meta-DNA.
 14. The method according to claim 10wherein the detecting step comprises using a detector comprising a CCD,EMCCD, EB CCD, CMOS, APS, or PMT.
 15. The method according to claim 10,wherein the waveguides are optical fibers or rectangular waveguides. 16.The method according to claim 10 wherein the probe array is a nucleicacid or protein probe array.
 17. A system to image the surface of asupport comprising: a source of excitation light; a first waveguidecapable of receiving light from the excitation light and transmittingthe light out an exit port; a second waveguide capable of receivinglight from an excitation light and producing an orientation of aninterference pattern when proximately positioned near the firstwaveguide, the second waveguide is capable of transmitting the light outan exit port; a rotating mechanism, wherein the rotating mechanismrotates at least one waveguide about an axis relative to the opticalpath to alter the orientation of the interference pattern; a supporthaving probes on a surface of a support facing the exit ports of thefirst and second waveguides; a detector capable of receiving reflectedsignals from the surface of the support; and a processor for analyzingthe detected signals.
 18. The system according to claim 17, wherein therotating mechanism further comprises rotating the first and secondwaveguides about a central axis to alter the orientation.
 19. The systemaccording to claim 17, further comprising altering a phase relationshipbetween the light in each optical path respective to each other, whereinan optical cladding is applied to at least one of the waveguides. 20.The system according to claim 17 wherein the probe array has abiopolymer affixed to the surface of a support selected from the groupconsisting of nucleic acids, oligonucleotides, amino acids, proteins,peptides, hormones, oligosaccharides, lipids, glycolipids,lipopolysaccharides, phospholipids, inverted nucleotides, peptidenucleic acids, and Meta-DNA.
 21. The system according to claim 17wherein the probe array is a nucleic acid or protein probe array. 22.The system according to claim 17, wherein the detector images the probearray.
 23. The system in accordance with claim 22, wherein the detectorcomprises a CCD, EMCCD, EB CCD, CMOS, APS, or PMT.
 24. The systemaccording to claim 17 further comprising a computer system to processthe detected image.
 25. The system according to claim 17 wherein thewaveguides are optical fibers or rectangular waveguides.
 26. A systemaccording to claim 17 further comprising: a dispersion cone in anoperative relationship with the exit port of the first waveguide fordispersing the exit light from the first waveguide; and a dispersioncone in an operative relationship with the exit port of the secondwaveguide for dispersing the exit light from the first waveguide.